Project Summary Combination antiretroviral therapy (cART) has transformed HIV-1 infection, once a fatal illness, to a manageable chronic condition. The latest cART regimen uses several classes of antiviral therapeutics and a typical therapy requires a combination of three or more drugs from at least two classes. Drug resistance, severe side effects and difficulties in treatment compliance have brought challenges to the implementation of cART in clinical settings and indicate the need for additional molecular targets. The first critical step of HIV-1 infection is fusion of viral and target cell membranes mediated by viral envelope glycoprotein (Env). Enfuvirtide is the first and still the only fusion inhibitor approved by FDA, but its limitations have restricted its long-term use. Moreover, the current therapies are not curative since they cannot eliminate latent HIV-1 reservoirs harboring integrated proviruses. Antibody-based therapeutics that can potently inhibit HIV-1 entry and also facilitate killing of Env-expressing cells are promising candidates for reservoir-eliminating strategies. The membrane proximal external region (MPER) of HIV-1 Env is a particularly attractive target for fusion inhibitors because it is one of the most conserved and functionally critical regions of the entire HIV-1 Env. Moreover, we have identified several MPER-directed hit compounds that can specifically inhibit membrane fusion mediated by HIV-1 Env but not SIV Env. Furthermore, the MPER-specific bnAbs exhibit extraordinary breadth in blocking HIV-1 infection and they are excellent candidates for developing therapeutic antibodies. In this project, we hypothesize that the MPER of HIV-1 Env is a promising therapeutic target for developing small-molecule and antibody-based inhibitors of viral membrane fusion. We will bring recent advances from the vaccine side of HIV-1 research into the search for novel therapeutics. We will purse the following specific aims: 1) we will identify small-molecule fusion inhibitors targeting the MPER of HIV-1 Env; 2) we will optimize small-molecule fusion inhibitors targeting the MPER; 3) we will develop antibody-based therapeutics targeting the MPER. When the project is completed, it will not only yield leads for preclinical testing, but should also provide novel reagents for further dissecting molecular mechanisms of HIV-1 entry.